Compositions and methods for treating cocaine-related disorders

ABSTRACT

Methods for treating a cocaine-related disorder in an individual include administering to the individual a therapeutic amount of an antibody comprising a human immunoglobulin gamma heavy chain and a murine lambda light chain. In another embodiment, the light chain includes a human kappa light chain at least partially derived from 1B3. Other embodiments are directed toward the antibodies themselves and methods of binding the antibodies.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/793,604, filed Apr. 20, 2006.

FIELD OF THE INVENTION

The present invention is directed to methods and compositions relatingto monoclonal antibodies.

BACKGROUND OF THE INVENTION

Cocaine is a powerfully addictive stimulant that directly affects thebrain. Cocaine, however, is not a new drug. In fact, it is one of theoldest known drugs. The pure chemical, cocaine hydrochloride, has beenan abused substance for more than 100 years, and coca leaves, the sourceof cocaine, have been ingested for thousands of years.

Today, cocaine use ranges from occasional use to repeated or compulsiveuse, with a variety of patterns between these extremes. There is no safeway to use cocaine and any route of administration can lead toabsorption of toxic amounts of cocaine, leading to acute cardiovascularor cerebrovascular emergencies that could result in sudden death.Repeated cocaine use by any route of administration can producedependence, addiction and other adverse health consequences.

Despite decades of basic and clinical research there are currently nomedications available to treat cocaine dependence, addiction, overdoseor to help prevent relapse. Thus, therapies are needed which can treatsuch cocaine-related disorders.

SUMMARY OF THE INVENTION

One embodiment of the present invention is directed toward a method fortreating a cocaine-related disorder in an individual. The methodincludes administering to the individual a therapeutic amount of anantibody comprising a human gamma heavy chain and a murine lamda lightchain.

In another embodiment, the present invention is directed toward a methodfor treating a cocaine-related disorder in an individual. The methodcomprises administering a therapeutic amount of an antibody comprising ahuman gamma heavy chain and a human kappa light chain at least partiallyderived from 1B3.

In another embodiment, the present invention is directed toward amonoclonal antibody comprising a human gamma heavy chain and a murinelambda light chain.

In another embodiment, the present invention is directed toward amonoclonal antibody comprising a human gamma heavy chain and a humankappa light chain at least partially derived from 1B3.

In an additional embodiment, the present invention is directed toward amethod for binding cocaine or a derivative thereof. The method includescontacting cocaine or a derivative thereof with an effective amount ofan antibody, wherein the antibody comprises a human gamma heavy gammachain and a light chain, wherein the light chain is selected from thegroup consisting of: a murine lambda light chain, a human kappa lightchain derived at least partially from 1B3, and combinations thereof.

These an additional embodiments of the invention will be more fullyapparent in view of the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description will be more fully understood in viewof the drawings in which:

FIG. 1 is a graph which depicts pharmacokinetics of an anti-cocaine mAb2E2 where mice receive an i.v. infusion of 120 mg/kg of 2E2, samples ofblood (10 μl) are obtained from tail veins at the indicated times afterthe completion of the mAb infusion, and concentrations of 2E2 in bloodare determined using an ELISA; data points represent the mean±SEM from 8mice, the Vdss is approximately 0.28 1/kg and a single compartment modelwith a t_(1/2) of 8.1 days adequately describes the elimination phase,represented by the best-fit regression line through the data points;

FIG. 2 is a graph which depicts the dose-dependent effect of 2E2 onplasma (A) and brain (B) concentrations of cocaine where mice areinjected with vehicle or 2E2 at doses of 12, 24, 40, 120 or 360 mg/kg;one hour after the infusion of vehicle or 2E2 is completed an i.v. bolusof cocaine HCl is administered and after five minutes the samples arecollected, cocaine concentrations are measured using GC/MS, symbolsrepresent the mean±SEM from three mice, the line through the data pointsrepresents the best fit according to a hyperbolic function, and theED₅₀s of 2E2 for decreasing the cocaine concentration in the brain andincreasing the plasma cocaine concentration are approximately 50 and 60mg/kg, respectively;

FIG. 3 is a graph which depicts the effect of 2E2 on plasma and brainconcentrations of cocaine metabolites where the concentrations of BE andEME are measured in the same tissue samples used for FIG. 2; plasma andbrain concentrations of BE are represented by closed and open squares,respectively, and plasma and brain concentrations of EME are representedby closed and open triangles, respectively; and

FIG. 4 is a graph which depicts the effect of 2E2 on thepharmacokinetics of cocaine in plasma (A) and brain (B) where micereceive an i.v. infusion of 120 mg/kg of 2E2, one hour later the micereceive an i.v. injection of cocaine HCl (0.56 mg/kg), and the animalsare sacrificed at the indicated times and samples are collected; thecocaine concentrations are determined by GC/MS, the data pointsrepresent the mean±SEM from three mice and in the absence of 2E2 (opencircles), the cocaine concentration-time profile in plasma (A) isdescribed by a two-compartment pharmacokinetic model with a t_(1/2α) of1.9 min and a t_(1/2β) of 26.1 min, while in the presence of 2E2 (closedcircles) a single compartment pharmacokinetic model indicated a t_(1/2)of 17.1 min, in the brain (B) in the absence of 2E2 (open circles) atwo-compartment pharmacokinetic model with first order input into thefirst compartment describes the cocaine concentration-time profile andthe calculated input t_(1/2) is 2.0 min and the t_(1/2α) and t_(1/2β)values are 2.0 min and 14.5 min, respectively, and in the presence of2E2 (closed circles), a single compartment pharmacokinetic model withfirst order elimination of cocaine indicates an elimination t_(1/2)value of 3.8 min.

The embodiments set forth in the drawings are illustrative in nature andare not intended to be limiting of the invention defined by the claims.Moreover, individual features of the drawings and the invention will bemore fully apparent and understood in view of the detailed description.

DETAILED DESCRIPTION

As used herein, “cocaine-related disorders” include cocaine dependence,addition, overdoe and/or relapse, and any other disorder resulting inwhole or in part from cocaine use. As the site of action of cocaine isin the brain, decreasing the concentrations reaching the brain would beexpected to decrease the probability of dependence, addiction, overdose,and relapse. Antibodies with high affinity and specificity for cocainewould be expected to act as pharmacokinetic antagonists by sequesteringcocaine in the peripheral circulation and preventing its entry to thebrain. Indeed, active immunization of animals with hapten-carrierconjugates can elicit the production of polyclonal anti-cocaineantibodies with sufficient levels and affinity for cocaine that they canreduce the amount of cocaine entering the brain. Active immunization hasalso been shown to attenuate the behavioral effects and the primingeffect of systemically administered cocaine in rats. Furthermore, theability of active immunization to produce levels of polyclonalanti-cocaine antibodies in humans that were associated with a decreasein use of cocaine demonstrates the potential efficacy of immunotherapyfor cocaine abuse. Unfortunately, individuals with compromised immunesystems (like those who have clinically induced immunosuppression orthose who suffer from some sort of an infection) can often not beactively immunized due to the risks of developing a complication fromthe active immunization. Often, those individuals who are suffering fromcocaine-related disorders also have compromised immune systems.

An alternative to active immunization is passive immunization. Inpassive immunization, a pre-made antibody is given to the individual.While this process is usually short lasting (a few days or even a fewweeks), it is much safer and effective for those with compromised immunesystems. In addition, using a monoclonal antibody (mAb) with a definedaffinity, specificity and dose may be even more efficacious than activeimmunization. Indeed, passive immunization with non-human anti-cocainemAbs attenuates the behavioral effects of cocaine and thereforerepresents an alternative or adjunct to active immunization.

Previously, a murine anti-cocaine antibody (GNC92H2) was generated anddemonstrated to have in vivo efficacy in rat models of cocaineaddiction. Also, two catalytic murine anti-cocaine mAbs that aredesigned to reduce blood cocaine levels through its hydrolysis have beengenerated and characterized. Unfortunately, non-human sequenceanti-cocaine mAbs would be expected to elicit an immune response inhumans similar to that elicited by the murine mAb OKT-3 used forimmunosuppression for organ transplant procedures. This immune responsewill target and try to destroy the non-human mAB thus decreasing orneutralizing the long-term efficacy of such an immunotherapeutic agent.Furthermore, the antibody affinity for cocaine should also be a majordeterminant of clinical efficacy. Unfortunately, the affinities of thecatalytic mAbs for cocaine are reported to be approximately 220 μM and55-5,240 μM, while the affinity of the anti-cocaine mAb GNC92H2 isreported to be 200 nM. Therefore, a more efficacious and predominantlyhuman antibody is likely to decrease the probability of inducing aneutralizing immune system response. This theory led to the generationand characterization of a monoclonal antibodies which are at leastpartially generated in transgenic mice that produce human sequence mAbs.

In contrast to the affinities of the catalytic and non-human mABs, theaffinity of these new antibodies for cocaine is approximately 4 nM,which is considerably higher than that of other anti-cocaine mAbscurrently under study. Additionally, these antibodies have highspecificity for cocaine over the major metabolites of cocaine.Therefore, these antibodies have important physicochemical propertiesthat confer efficacy as a passive immunotherapeutic agent.

Therefore, according to one embodiment, the present invention isdirected toward a monoclonal antibody comprising a human gamma heavychain and a murine lamda light chain. In one embodiment, the murinelambda light chain comprises SEQ ID NO: 1 or a derivative thereof. Inanother embodiment, the human gamma heavy chain comprises SEQ ID NO: 2.In a further embodiment, the human gamma heavy chain comprises SEQ IDNO: 2 and the murine lambda light chain comprises SEQ ID NO: 1. In oneembodiment, the combination of these sequences is known as 2E2. Inanother embodiment, the antibody comprises a human gamma heavy chain anda human kappa light chain derived at least partially from 1B3. Inanother embodiment, the antibody is an immunoglobulin.

According to another embodiment, the invention is directed toward amethod of treating a cocaine-related disorder in an individual. Themethod includes administering a therapeutic amount of an antibody to theindividual including a human immunoglobulin gamma heavy chain and amurine lambda light chain. The human gamma heavy chain contains themajority of the specificity for cocaine and its derivatives and thus itmay be used in combination with any light chain which effects atherapeutic effect for cocaine-related disorders.

In one embodiment, the human gamma heavy chain comprises SEQ ID NO: 2 ora derivative thereof. In an additional embodiment, the light chaincomprises a murine lambda light chain including SEQ ID NO: 1 or aderivative thereof. In one embodiment, the murine lambda light chain ispartially murine derived. In further embodiment, the partially murinederived light chain comprises a murine variable region and a humanconstant region. Derivatives as employed herein include those sequenceswhich would be functionally equivalent to either chain of the antibody.This would include those antibodies which have the same primarystructure (i.e. sequence), but have a different tertiary structure dueto the addition of, for example, a salt or a sugar. In addition, thefunctional regions of the sequences have been identified as the variableregions. Thus, heavy and/or light chains including at least one variableregion and maintaining their ability to be therapeutic forcocaine-related disorders are also included. In the murine lambda lightchain, there are three variable regions. These regions reside at aminoacid residues numbered 23-36 (SEQ ID NO: 4), 52-58 (SEQ ID NO: 5), and91-99 (SEQ ID NO: 6). In the human gamma heavy chain, these regionsreside at amino acid residues numbered 32-37 (SEQ ID NO: 7), 51-67 (SEQID NO: 8), and 100-103 (SEQ ID NO: 9). In the human kappa light chain,these regions reside at amino acid residues numbered 24-34 (SEQ ID NO:10), 50-56 (SEQ ID NO: 11), and 89-98 (SEQ ID NOS: 12 and 13).

The variable regions or CDR regions are the sites for ligand-antibodyinteractions. Thus, in one embodiment, derivatives of SEQ ID NO: 1 maycomprise one or more of SEQ ID NOS: 4, 5, and 6 and derivatives of SEQID NO: 2 may comprise one or more of SEQ ID NOS: 7, 8, and 9.Additionally, derivatives of the variable regions could also be used,for example derivatives having between 90-95%, 85-90%, 80-85%, 75-80%,70-75%, 65-70%, or 60-65% homology to their respective variable regionsequences. The resulting antibodies from the derivatives of the variableregions also have the same cocaine binding functionality as the otherdisclosed antibodies.

In an additional embodiment, a method for treating cocaine-relateddisorders includes administering a therapeutic amount of an antibodycomprising a human immunoglobulin gamma heavy chain and a human kappalight chain at least partially derived from 1B3 to an individual. Thislight chain is selected because this sequence has been shown bysequencing of genomic DNA to be generated by light chain generecombination in the hybridoma cell line 2E2 that produces mAb 2E2. Theanti-digoxin mAb 1B3 is a fully human sequence mAb that binds digoxinand digitoxin with high affinity (nMolar). This antibody has beendescribed in two publications. Farr, C. et al., “Three-dimensionalQuantitative Structure-Activity Relationship Analysis of Human SequenceAntidigoxin mAbs using CoMFA.” Journal Medicinal Chemistry 45 (15):3257-3270, 2002 and Paul, S., Monson, N. & Ball, W. J., “MolecularModeling of Cardiac Glycoside Binding by the Human sequence mAb 1B3.”Proteins: Structure, Function and Bioinform. 60: 382-391, 2005,incorporated herein by reference. In one embodiment, the human lightkappa chain comprises SEQ ID NO: 3 (1B3a) or a derivative thereof. Inanother embodiment, the human kappa light chain comprises SEQ ID NO: 14(1B3b) or a derivative thereof. In one embodiment, derivatives of SEQ IDNOS: 3 and 14 may comprise one or more of SEQ ID NOS: 10-13.Additionally, derivatives of their CDR regions are also applicable asdescribed above for SEQ ID NOS: 1 and 2.

In another embodiment, an antibody binds cocaine or a derivativethereof. Radioligand binding assays using an antibody and [³H]-cocaineyields an average dissociation constant (K_(d)) of 4.4 nM. Bindinginhibition constants (K_(i)) of the cocaine derivatives are determinedby competition assays with a constant [³H]-cocaine concentration andvarying concentrations of nonradioactive competitors. The results showthat the present antibodies bind cocaethylene with an affinity(K_(i)=3.4 nM) nearly identical to that for cocaine. On the other hand,the affinities for the three physiologically important but inactivecocaine derivatives benzoylecgonine, ecgonine methyl ester, andecogonine are significantly lower (K_(i) values of 43 nM, 5.2 μM and 95μM, respectively), revealing the importance of the benzoyl moiety forhigh binding affinity. Even K_(d) values of 40 nM show beneficialresults. Other examples of cocaine derivatives include: (−) cocaine;cocaine propyl ester; RTI-128; RTI-66; RTI-160; RTI-192;m-hydroxycocaine; WIN 35,065-2; WIN 35,428; RTI-31; RTI-32; RTI-55;RTI-111; m-hydroxybenzoylecgonine; p-hydroxybenzoylecgonine; RTI-113;tropine; benztropine; 4′,4″-difluoro-3α-diphenylmethoxytropane;hyoscyamine-N-oxide; methylanisotropine; tropisetronmethiodide;anisodine; scopotamine; scopotamine-N-oxide; methylscopolamine;N-butylscopolamine; (−) pseudococaine; (+) cocaine; norcaine;benzoylnorecgonine; (+) pseudococaine; ecgonidine;exo-6-hydroxytropinone; and methylcocaethylene.

The monoclonal antibodies of the present invention use their bindingaffinity for cocaine and its derivatives to reduce the concentration ofcocaine or its derivatives in the brain. Infused antibodies also producea dramatic dose-dependent increase in plasma cocaine concentrations anda concomitant decrease in the brain cocaine concentrations produced byan i.v. injection of cocaine HCI (0.56 mg/kg). At the highest dose ofantibody tested (3:1, mAb:drug), cocaine is not detectable in the brain.Pharmacokinetic studies show that the normal disappearance of cocainefrom plasma is described by a 2-compartment pharmacokinetic model withdistribution t_(1/2α) and terminal elimination t_(1/2β) values of 1.9and 26.1 min, respectively. In the presence of an equimolar dose of mAb2E2 there is a 26-fold increase in the area under the plasma cocaineconcentration-time curve (AUC) relative to the AUC in the absence of2E2. Consequently, the antibodies of the present invention decreasecocaine's volume of distribution from 6.0 l/kg to 0.20 l/kg, whichapproximates that of 2E2 (0.28 l/kg). However, cocaine is still rapidlycleared from plasma and its elimination is now described by a singlecompartment model with an elimination t_(1/2) of 17 min. Importantly,the antibodies also produce a 4.5-fold (78%) decrease in the cocaine AUCin the brain. Therefore, the effect of the antibodies on plasma andbrain cocaine concentrations is predominantly due to a change in thedistribution of cocaine with negligible effects on its rate ofclearance.

In addition to being a monotherapy, embodiments also include additionalco-therapies. For example, when treating someone in rehabilitation toprevent relapse, an antibody according to the invention can beadministered in conjunction with treatments for withdrawal symptoms (forexample, administration of amantadine and propranolol). An antibodyaccording to the invention can be used in conjunction with counselingand other forms of psychotherapy. In addition, it can be used with anyantagonist or agonist pharmacotherapies that use compounds for which theantibody does not have substantial affinity. The antibody can also beused with other antibodies that target other drugs of abuse ormedicaments.

An antibody according to the present invention may be administered byany suitable route or device. In one embodiment, the antagonist will beadministered by injection. The most common form of delivery will be anintravenous injection or infusion.

The antibody is administered in an amount sufficient to treat thecocaine-related disorder. The treatment as used herein encompasses areduction in clinical symptoms of the disorder and/or elimination of thedisorder. Therapeutic amounts will vary based on an individual's age,body weight, symptoms, and the like, and may be determined by one ofskill in the art in view of the present disclosure. Initial clinicalstudies of a cocaine vaccine do provide vital information about theconcentrations of anti-cocaine antibodies required to decrease cocaineuse by cocaine abusers. In vaccinated patients the highest mean serumantibody titer would correspond to about 61.4 μg/mL, and a decrease incocaine use is reported in this cohort as well as cohorts with lowermean antibody titers. As the standard blood volume in a 70 kg person is2.8 liters, then the quantity of anti-cocaine antibodies that conferefficacy in patients is about 61.4 μg/mL×2,800 ml or about 172 mg/person(about 2.5 mg/kg). This is likely be a minimally effective dose. Bycomparison, doses of 40 and 120 mg/kg can be safely administered and areefficacious in rodent models. These doses translate to 2,800-8,400 mg ina 70 kg person, almost 20-50-fold higher than may be required to conferefficacy in humans. Additionally, it should be noted that the polyclonalanti-cocaine antibodies that constituted the standard immune response invaccinated patients had an average affinity (Kd) of 28 nM [17], whilethe present anti-cocaine monoclonal antibodies have a higher affinity(for example, 2E2 has a Kd=4 nM). Therefore, equimolar doses of 2E2 aremore effective or equieffective doses would be lower.

In addition to its use as a treatment for cocaine-related disorders, theantibodies of the current invention could also be used in screeningassays for the development of other therapeutics, including othertherapeutics useful for the treatment of cocaine. Thus, one embodimentof the present invention also includes a method for binding cocaine or aderivative thereof. The method comprises contacting cocaine or aderivative thereof with an effective amount of an antibody. The antibodycomprises a human gamma heavy chain and a light chain. In oneembodiment, the light chain is selected from the group consisting of: amurine lambda light chain, a human kappa light chain at least partiallyderived from 1B3, and combinations thereof. Additionally, as describedabove, the heavy and light chains can be several different combinationsand/or variations.

The following examples demonstrate various specific embodiments of theinvention.

EXAMPLES Methods

Animals. Jugular vein catheterized male Swiss-Webster mice (22-28 g atthe start of the studies) are purchased. Mice are housed individuallywith free access to food and water and kept on a 12 h light/dark cycle.These studies are carried out in accordance with the Guide for the Careand Use of Laboratory Animals under a protocol approved by theInstitutional Animal Care and Use Committee at the College of Medicine,University of Cincinnati.

Cocaine pharmacokinetic studies. Prior to the start of the studies, thepatency of the venous catheters is verified by demonstrating the abilityto withdraw blood or inject normal saline via the catheter. The antibody(3-5 mg/ml) in phosphate-buffered saline (PBS) or vehicle (PBS) isinfused at a rate of approximately 0.35 ml/min for up to two min,depending on the antibody concentration and the body weight of theanimal, with the animal held under mild restraint. One hour aftercompletion of the infusion of mAb, cocaine HCl (0.56 mg/kg) plus heparin(400 units/kg) is injected intravenously through the same catheter at avolume of 4.0 ml/kg body weight. At most sampling times, sodiumpentobarbital (45 mg/kg, i.p) is administered three minutes prior tosacrificing the animal. For the 0.75 min time point the cocaine isinjected into anesthetized mice. At the designated times after theinjection of cocaine, anesthetized mice are sacrificed by decapitationand trunk blood (typically 0.8-1.2 ml) is collected in a 1.5 mlpolypropylene microcentrifuge tube containing 11.2 μl heparin (1.0unit/μl) and NaF (16 mg/0.8 ml of blood) to inhibit, respectively, bloodcoagulation and enzymatic hydrolysis of cocaine. The blood samples arecentrifuged at 5,000×g for 3 min, then the plasma (typically 0.4-0.8 ml)is carefully separated from packed red blood cells, placed into sterile1.5 ml Eppendorf microcentrifuge tubes, rapidly frozen on dry ice andthen stored at −80° C. until analysis.

At the same time a separate sample of blood (approximately 100 μl) iscollected from each mouse and rapidly frozen on dry ice then stored at−80° C. The concentration of hemoglobin and, where appropriate 2E2, ismeasured in these samples.

The whole brain is quickly removed from the decapitated mice, surfaceblood is blotted away, and the brain is placed in a polypropylene tube,rapidly frozen on dry ice and then stored at −80° C. until analysis. Foranalysis, brains are weighed and cold deionized, distilled water addedto produce a total volume of 1 ml, then homogenized and centrifuged at13,000 rpm for 45 min at 4° C. The resulting supernatants (0.4-0.6 ml)are collected into sterile polypropylene microcentrifuge tubes and analiquot (0.05-0.40 ml) is processed for cocaine/metabolite analysis byGC/MS and hemoglobin content. Any remaining sample is stored at −80° C.

Determination of blood and brain hemoglobin concentrations. Thehemoglobin contents of brain and blood are quantified spectroscopicallyby combining the method reported by Choudhri et al. (1997) and aprotocol provided by Pointe Scientific, Inc. (MI). In this procedure, 10μl aliquots of blood or 50 μl aliquots of brain homogenate supernatantsare diluted with 90 μl hemoglobin reagent (0.6 mM K₃Fe(CN)_(6,) 0.7 mMKCN) in glass test tubes. The reaction is allowed to proceed at roomtemperature for 15 min with gentle mixing. When the reaction iscomplete, aliquots from the standards and samples are all transferredinto PVC microtiter plate wells and the absorbance is measured at awavelength of 490 nm for the measurement of cyanmethemoglobin formation.For the similarly prepared hemoglobin standards, the absorbance isdirectly proportional to the hemoglobin concentration over the rangeused (0.3-12 g/dl). The standard curve is verified using controlstandards and the hemoglobin concentration in each sample is determinedby comparison with the standard curve. The mean±SEM concentration ofhemoglobin in whole blood and brain are determined to be 8.90±0.32 g/dland 0.22±0.04 g/dl, respectively. The average hemoglobin content inbrain tissue relative to that present in whole blood is, therefore,approximately 2.5%.

2E2 in vivo pharmacokinetic studies: Sample preparation. Mice, whileunder mild restraint, are administered mAb 2E2 (120 mg/kg, at 4.2 mg/mlin PBS) via an intravenous infusion over a 2 min period. Then at varyingtimes, to obtain blood samples for mAb quantification, the mice areanesthetized using isoflurane and a sterile 27-gauage hypodermic needleor, alternatively, a sterile scalpel blade is used to puncture or make asmall cut in a tail vein and 10 μl of blood is collected using aheparinized capillary pipette tip. The blood is immediately placed in a1.5 ml polypropylene microcentrifuge tube containing 40 μl of 0.1 Msodium citrate/0.1% sodium azide pH 4.75. These samples are then rapidlyplaced on ice and then stored at 4° C. until use. A blood sample istaken immediately prior to the infusion of 2E2 and then at 3, 15 and 30min, 1, 2, 4 and 8 hr, 1 day and periodically up to 49 days as shown inthe results.

mAb 2E2 quantification: ELISA. The in vivo concentrations of 2E2 aredetermined using an enzyme-linked immunosorbent assay (ELISA) thatcompares the quantity of mAb in varying dilutions of the mouse bloodsamples to that quantified in a standard curve generated using knowndilutions of purified 2E2 or human IgG. Briefly, the conjugatebenzoylecgonine-ovalbumin (3 μg/ml, 100 μl/well) in 1 mM EGTA pH 7.4 isadsorbed onto PVC 96-well microtiter plates for 1 hr. The plates arethen washed 3 times with, and all wells exposed for 10 min to, 0.5% BSAin TBS (10 mM Tris, 140 mM NaCl and 0.02% NaN₃, pH 6.9) in order toblock non-specific protein binding. The second layer, 100 μl/well of theblood samples diluted (1:500) into BSA-TBS, is added and the sample isincubated for 2 hr. Serving as quantitation standards, duplicate 100μl/well samples of human IgG or 2E2 diluted over a range ofconcentrations from 0.003-3.0 μg/ml are also similarly plated andincubated. The plates are washed with a Solution A, containing 0.5% BSA,10 mM sodium phosphate, 145 mM NaCl, 1.5 mM MgCl_(2,) 0.05% triton X-100and 0.02% NaN₃, pH 7.2. Then 50 μl/well of affinity-purifiedbiotinylated goat anti-human IgG diluted 1:500 in Solution A is addedand incubated for 1 hr. After washing, 50 μl/well ofstreptavidin-alkaline phosphatase, diluted (1:200) in Solution A, isadded, incubated for 1 hr and removed. Then 50 μl/well of thecolorimetric reaction mixture, comprised of the substratepara-nitrophenylphosphate (1 mg/ml) in substrate buffer (50 mMNa₂CO_(3,) 50 mM NaHCO₃ 1 mM MgCl₂ at pH 9.8), is added. After 6-8 minthe reaction is stopped with 1M sodium hydroxide (50 μl/well). All stepsare performed at room temperature. The reaction endpoint is measuredwith an ELISA reader at a wavelength of 405 nm. Each determination isdone in duplicate.

Antibodies: The hybridoma cell line secreting mAb 2E2 is generated usingstandard hybridoma technology by fusing spleenocyctes obtained from atransgenic mouse, strain HCo7/Ko5, following its immunization withbenzoylecgonine (BE) coupled to 1,4-butanediamine-derivatized keyholelimpet hemocyanin (KLH) with the mouse cell line P3X63-Ag8.653.Production of mAb 2E2 is accomplished by growing hybridomas in severecombined immunodeficient (SCID) mice and collecting the ascites fluid.The hybridoma-secreted mAb is purified from ascites by sodium sulfateprecipitation and a several step protein A-Sepharose columnchromatography procedure adapted from that previously described.Identification of the full length amino acid sequences of thepolyacrylamide gel separated heavy and light chains of the 2E2 moleculeis accomplished using liquid chromatography/mass spectroscopy (LC/MS/MS)analysis of their tryptic fragments. The heavy (H) chain is identifiedas a γ₁ protein of the human VH3 family gene DP-50. The light (L) chainis identified as a mouse λ VL2. The MS sequencing is consistent with andconfirmed results obtained previously from Edman degradationNH₂-terminal sequencing of the Western blotted H and L chains as well asthe sequencing of mRNA-dependent cDNA representing the 2E2, V_(H) andV_(L) chain regions. The γ1 human H chain NH₂-terminal sequence is:EVQLVESGGGLVKPGGSLRL-(see SEQ ID NO: 2), while the mouse λ chainNH₂-terminal sequence is: QAVVT/IQESALTTSPGGTV-(see SEQ ID NO: 1).Although the 2E2 hybridoma contains the recombined DNA sequence for ahuman κ L6 light chain, and this is consistent with the human κ chainsof anti-digoxin antibodies generated from these transgenic mice inprevious work, the L chain for the mAb expressed and used is a murine λ.These results are consistent with a recent report that hybridomas fromthe HCo7/Ko5 strain of transgenic mouse can generate mixed-chain, humanH, mouse L mAbs in addition to human sequence mAbs. Overall, 2E2 hasabout an 87% sequence identity/homology with human IgG(λ)1immunoglobulins.

The murine anti-cocaine mAb 3P1A6 obtained from BioDesign International,Inc., (Saco, ME) has previously been reported to have a high affinity(K_(d)=0.2 nM) for cocaine and approximately 12-fold and 1,500-foldlower affinities for the inactive metabolites benzoylecgonine (BE) andecgoninemethylester (EME), respectively. The murine anti-cocaine mAbB4E10 has been determined to have a moderate affinity for cocaine(K_(d)=40 nM) and approximately 30-fold and 50,000-fold lower affinitiesfor BE and EME, respectively. Therefore, the murine mAbs and 2E2 havesimilar specificities for cocaine over its major metabolites, but anapproximately 200-fold range difference for cocaine affinity. As anadditional control, to test for non-specific in vivo effects resultingfrom infusion of mAb, non-specific human polyclonal IgG immunoglobulinis administered to mice. These latter immunoglobulins have no measurableaffinity for cocaine or its major metabolites (data not shown).

Solid Phase Extraction of cocaine and metabolites from plasma and brain.In order to determine in vivo concentrations of cocaine and itsmetabolites BE and EME, following the i.v. injection of cocaine, 100-400μl samples of heparinized/NaF treated plasma and 400 μl samples of brainhomogenates obtained from cocaine-treated animals are added to 2 ml of0.1 M Na phosphate buffer, pH 6.0. This is followed by the addition of5% trichloroacetic acid at a volume equal to that of the experimentalsample (100-400 μl). These mixtures are shaken for 20 minutes and thencentrifuged for 15 min at 7000 rpm, all at room temperature, in order toprecipitate the denatured protein. The supernatants are collected andadjusted to pH 5.4 with 10 M NaOH. Then to serve as internal standardsfor establishing the identification of cocaine and its metabolites aswell as for normalization of the recovery of cocaine/metabolites fromthe mouse samples, 50 μl of a sample containing deuterated (D₃)cocaine-D₃, BE-D₃ and EME-D₃ (each at 1 μg/ml) is added to all of theexperimental and the standard control samples before their undergoingsolid-phase extraction/column elution. Duplicate, standard control tubes(2 ml) are also prepared containing; 0.1 M Na phosphate buffer, 50 μl ofthe internal standards D₃ cocaine/BE/EME (1 μg/ml, each), 200 μl ofnormal mouse plasma and varying amounts of cocaine (1-500 ng) and usedto generate the standard cocaine concentration curves. Similarly,standard concentration curves are also generated for BE and EME. Also 10μl of the stock solution of cocaine HCl (0.139 mg/ml) that is infusedinto the mice is also mixed with the phosphate buffer and thecocaine-D₃/metabolite-D₃ standards for quantification of the cocaineadministered to the animals. Thus, the cocaine/metabolite levels aredetermined relative to that of standard samples undergoing the samecolumn extraction, elution and derivatization procedures.

The procedure of Varian is used to extract and recovercocaine/metabolites from the plasma and brain samples and standards.First, Bond Elut Certify columns with the non-polar C8 sorbent, set in aVarian vacuum manifold are conditioned by washing with 2 ml methanol,followed by 2 ml of 0.1 M Na phosphate buffer, pH 6.0. Next, theprepared plasma and brain homogenate samples (2 ml) are loaded onto theBond Elut columns. The columns are then washed with 6 ml of deionizedwater, 3 ml of 0.1 M HCl, and 9 ml of methanol. The column-boundanalytes are then eluted with 2-3 ml of a freshly prepared solution ofdichloromethane:2-propanol:ammonium hydroxide (mixed: 78:20:2, v/v/v).These extracts are then evaporated to dryness under nitrogen at 45° C.for 15 min. The residue samples are derivatized with 25 μlN-methyl-N-trimethylsilyl trifluoroacetamide (MSTFA) mixed with 25 μlethyl acetate. These samples are vortexed and incubated at 65° C. for 30min. After cooling, the trimethylsilyl-derivatized samples aretransferred to glass autosampler vials for analysis by GC/MS. The GC/MSanalysis of analytes is typically completed within 1-2 hours of samplederivatization. Analyses carried out more than eight hours afterderivatization are discarded.

Gas chromatography/mass spectrometry. The gas chromatograph/massspectrometer (GC/MS) consisted of a Shimadzu GC 17A series GC,interfaced with a Shimadzu QP-5050A quadruple MS fixed in an electronimpact ionization mode with selective ion monitoring. The GC/MS isoperated with a transfer line temperature of 280° C. and a sourcetemperature of 280° C. The MS is calibrated on a daily basis usingperfluorotributylamine. The electron multiplier voltage is set at 1.2kV. Chromatographic separation is achieved using a Restek Rtx-5MS crosslinked 5% diphenyl-, 95% dimethylsiloxane capillary column (30 m×0.25 mmi.d, 0.25 μm film thickness). Helium is the carrier gas and used at aflow rate of 1.0 ml/min.

A Shimadzu AOCs autosampler is used to inject 2 μl of extract sampleinto the GC/MS. The GC, equipped with split/splitless injection port, isoperated at 280° C. in the splitless mode with a high pressure injectionset at 150 kPa for 0.75 min. The oven temperature profile is establishedas follows: the initial temperature is 100° C. and it is held for 1 min,then increased at a rate of 20° C./min up to 320° C. This temperature isheld for 8 min resulting in a total run time of 20 min. The lower limitsof cocaine/BE/EME detection ranged from 1-5 ng/ml and the linear dynamicrange for most analytes is 1-3000 ng/ml. The instrument performance isevaluated by analysis of the calibrator and control samples. Analytesare identified and their concentrations are determined using both theinternal deuterated standards and concentration control samples preparedwith normal mouse serum, respectively, as described above. The responsefactor is determined for each analyte. The response factor is calculatedby dividing the area of the analyte peak by the area of the internalstandard peak. Calibration curves are then prepared by plotting a linearregression of the analyte/internal standard response factor versus theanalyte concentration for all calibrators analyzed. The standard curveis constructed using a set of cocaine/metabolite samples varying over aconcentration range of 1-500 ng/ml. The standard curve is used todetermine concentrations of analytes in both control and experimentalsamples.

Chemicals reagents and reference standards: Standard solutions ofcocaine, BE and EME (each 1 mg/ml) are prepared in methanol oracetonitrile and serve as stock solutions for preparing the referencestandard curves. The cocaine-D₃, BE-D₃ and EME-D₃ are used as theinternal standards (0.1 mg/ml each in methanol or acetonitrile). MSTFAis the derivatizing reagent. Normal mouse plasma with heparin isobtained. The human hemoglobin standards and control standards areobtained. All other chemicals and immunoreagents are purchased. Allreagents and organic solvents are of analytical grade or HPLC grade.

Data analysis and statistics: Cocaine and 2E2 pharmacokinetic data areanalyzed using the program WinNonLin. The program provides AkaikeInformation Criterion (AIC) and Schwartz Bayesian Criterion (SBC)measures of “goodness of fit” of the data to the one or two compartmentpharmacokinetic models that are used. Data are first analyzed accordingto a single compartment pharmacokinetic model. In some experiments asingle compartment model gave a poor fit to the cocaine pharmacokineticdata, as assessed by a systematic deviation of the model from the data.In these cases the fit to the data is improved by applying a twocompartment pharmacokinetic model that assumes cocaine distributedbetween a central and a peripheral compartment. In addition to animprovement in the AIC and SBC measures, the improvement of the fit ofthe model to the data is evaluated by a lack of a systematic deviationfrom the data points and a concomitant reduction in the sum of squaresresiduals. Applying pharmacokinetic models that assumed that cocainedistributed between more than two compartments only slightly improvedthe fit to the observed data and this additional complexity isconsidered unnecessary. Statistical comparisons of the cocaine andmetabolite levels observed in the presence and absence (vehicle) ofantibody at the single 5 min time point used non-parametric Mann-Whitneytest while the Analysis of Variance (ANOVA) procedure is used to comparethe results obtained over different experimental days.

Results

The pharmacokinetics of mAb 2E2. In determining the pharmacokinetics ofmAb 2E2 in mice, the first samples of tail vein blood are taken 3minutes after completion of the i.v. infusion of 2E2 (120 mg/kg) via thejugular vein of mice. The initial mean±SEM blood concentration of mAb isdetermined to be 370±17 μg/ml (n=8 mice). As shown in FIG. 1 there is noevidence for an initial decrease in blood concentrations over the first24 hours. Indeed, 2E2 concentrations increased slightly over the firstfour hours and then appeared to plateau for approximately 20 hours. Themean concentration of 2E2 as measured 24 hours after infusion is 422±21μg/ml. After 24 hours, the concentrations of 2E2 in blood then begin todecline and this is adequately described by a single compartmentpharmacokinetic model with an elimination t_(1/2) of 8.1 days (FIG. 1).This model gives a calculated volume of distribution at steady state(Vdss) for 2E2 in this group of mice of 0.28 l/kg.

The plasma pharmacokinetics of cocaine. Next, the disposition of cocainein mouse plasma subsequent to its i.v. injection via the jugular vein isdetermined. The highest plasma concentrations measured (˜110 ng/ml) areobserved at the earliest sample time, after which cocaine concentrationsdeclined rapidly (FIG. 4A). A pharmacokinetic model assumes that cocainedistributed between a central and a peripheral compartment improves thefit to the observed data as compared to a single compartment model. Thisresult is similar to that which has previously been reported for i.v.injected cocaine in several species including rats, non-human primatesand for i.p. injected cocaine in mice. The simplest pharmacokineticmodel that provides a general description of the data generatedparameter estimates for the distribution half-life (t_(1/2α)) andterminal elimination half-life (t_(1/2β)) for cocaine of 1.9 and 26.1min, respectively. The calculated Vdss is 6.0 l/kg.

Effect of cocaine-specific mAbs on cocaine distribution. In theseexperiments a single time point, 5 minutes after i.v. cocaineadministration, is selected at which to determine the effect ofcirculating anti-cocaine mAbs on the in vivo plasma and brain levels ofcocaine. As shown in Table 1 below, at 5 minutes considerabledistribution of cocaine has occurred as the plasma concentration (˜77ng/ml) declines from an initial value of ˜110 ng/ml (45 sec, FIG. 4A)and brain levels (˜1070 ng/g) are about 10-fold higher than that inplasma. The presence of mAb 2E2 then produced a substantial 29-foldincrease in plasma and an almost 5-fold decrease in brain cocaineconcentrations (Table 1) in comparison to the vehicle controls.Furthermore, pretreatment with the mouse anti-cocaine mAbs 3P1A6(K_(d)=0.2 nM) and B4E10 (K_(d)=40 nM,) also similarly increased cocaineconcentrations in plasma, while they are somewhat less effective than2E2 in decreasing cocaine concentrations in the brain. These resultsclearly demonstrate the capability of cocaine-specific mAbs for in vivobinding of cocaine. In contrast, the pretreatment with non-specifichuman polyclonal antibodies with no measurable affinity for cocaineproduced a small increase in cocaine concentrations in both plasma andbrain relative to those in mice pretreated with the vehicle (PBS). TABLE1 Change Change Plasma cocaine from Brain cocaine from concentration(ng/ml) vehicle concentration (ng/g) vehicle Vehicle 76.6 ± 3.3 (n = 23)1070.5 ± 32.1 (n = 22)  Human IgG 121.2 ± 5.6* (n = 6)   +1.6-fold  1568± 130.5* (n = 5) +1.5-fold 2E2 2197.7 ± 75.8** (n = 6) +28.7-fold 223.7± 25.5** (n = 6) −4.8-fold 3P1A6  2215.5 ± 157.2** (n = 6) +28.9-fold469.2 ± 68.9** (n = 6) −2.3-fold B4E10 1591.5 ± 57.8** (n = 6)+20.8-fold 560.5 ± 62.4** (n = 6) −1.9-fold

The dose-dependent effect of 2E2 on plasma and brain cocaineconcentrations. In view of the magnitude of the effects ofstoichiometric doses of the anti-cocaine mAbs on the plasma and braincocaine concentrations, the dose-dependency for the responses isdetermined using mAb 2E2. In the absence of 2E2 the mean±SEM plasmacocaine concentration at 5 min post cocaine injection is 78.5±4.5 ng/ml(FIG. 2A). Infused 2E2 produces a dose-dependent increase in plasmacocaine concentrations (FIG. 2A). The lowest dose of 2E2 (12 mg/kg, a1:10 mAb:cocaine ratio) produces a significant (p<0.01, one-way ANOVAwith post-hoc test) 5.1-fold increase in plasma cocaine concentrationswhile the highest dose (360 mg/kg, a 3:1 ratio) produces a dramatic46.1-fold increase in cocaine concentrations. The calculated dose of 2E2that produces 50% of the highest effect for the range of 2E2 doses used(ED₅₀) is approximately 80 mg/kg, a somewhat less than stoichiometricamount of 2E2.

In the absence of 2E2 the mean±SEM brain cocaine concentration at 5 minpost injection, corrected for cocaine present in cerebral blood, is796.8±50 ng/ml (FIG. 2B). This represents a brain:plasma cocaineconcentration ratio of 10:1. 2E2 then produces a dose-dependent decreasein brain cocaine concentrations (FIG. 2B). At the dose of 24 mg/kg, 2E2produces a significant 35% decrease in cocaine concentrations. At the2E2 dose of 360 mg/kg, after correction for cocaine present in theresidual blood, the brain cocaine concentration is negligible. The ED₅₀for the range of 2E2 doses used is approximately 60 mg/kg.

The effect of 2E2 on cocaine metabolite concentrations in plasma andbrain. An additional point of interest is the determination of theeffects of circulating 2E2 on the in vivo metabolism of cocaine. Asshown in FIG. 3, at 5 min after the injection of cocaine in the absenceof 2E2, mean concentrations of the predominant cocaine metabolite inmice, EME, which results largely from plasma butyrylcholinesteraseactivity, are 25 ng/ml and 267 ng/g in plasma and brain, respectively.This represented a brain:plasma ratio for EME of 10.7:1, a ratio similarto that of cocaine in these mice (FIG. 2), thus the cocaine:EME ratio is˜3:1 in both plasma and brain. Produced by non-specific livercarboxylesterase activity, BE levels are lower with mean concentrationsof 7 ng/ml and 16 ng/g in plasma and brain, respectively, representing abrain:plasma ratio of 2.3:1. A modest increase (˜3-fold) in plasma BEconcentrations is observed with increasing doses of 2E2 but the effectapproached a plateau at 2E2 doses above 40 mg/kg. There is a concomitantdecrease in brain BE concentrations which is observed at doses above 40mg/kg. These results are consistent with mAb 2E2 having no effect on BEproduction but a sufficiently high affinity for BE to sequester some inthe plasma, but its levels are limited. In contrast, plasma EMEconcentrations appear unaffected at the lower doses of 2E2, but anapproximate 2-fold reduction is observed at the highest dose of 2E2.There is no systematic effect of 2E2 dose on brain EME concentrations(FIG. 3). It is of note, that despite 2E2's effective in vivo binding ofcocaine, its alterations in cocaine's initial metabolism appear modest.

Effect of 2E2 on the pharmacokinetics of cocaine in plasma and brain.Next, the effects of a stoichiometric dose of 2E2 on thepharmacokinetics of a single injection of cocaine are determined. Asshown in FIG. 4A, in the presence of 2E2, the peak plasma concentrations(˜1,100 ng/ml) of cocaine are observed at the earliest time point (45sec) sampled after its injection. This is similar to what is observed inthe absence of 2E2. However, the peak plasma concentration is 11.3-foldhigher in the presence than in the absence of 2E2. Furthermore, incontrast to the biexponential decrease in the concentrations of cocaineobserved in the absence of 2E2, the decrease in the cocaineconcentration is well described by a pharmacokinetic model that assumesa single compartment and no initial distribution phase. Thus, thecalculated t_(1/2) for the disappearance of cocaine from plasma in thepresence of 2E2 is 17.1 min and this contrasts with the distribution andelimination phases, with parameter estimates for t_(1/2α) and t_(1/2β)of 1.9 and 26 min, respectively. 2E2 also produces a sustained increasein the plasma cocaine concentration that results in 26-fold increase inthe area under the concentration-time curve (AUC) in plasma. Consistentwith this result, the calculated Vdss of cocaine in the presence of 2E2is 0.2 l/kg as compared to 6.0 l/kg in the absence of 2E2.

As shown in FIG. 4B, the cocaine concentration-time profile in braindiffers substantially from that observed in the plasma (FIG. 3A). Theconcentration of cocaine in the brain (corrected for cocaine present inresidual blood) at 45 sec (˜650 ng/g) after the injection isapproximately 6-fold higher than that measured in plasma. The braincocaine concentrations subsequently increase further and the highestmeasured concentration is observed at 3 min (˜1,500 ng/g), after whichconcentrations then rapidly decline. A pharmacokinetic model thatassumes a first-order input to the brain and a first-order output isused to describe the increase and subsequent decrease in brain cocaineconcentrations. The estimated t_(1/2) for entry into the brain isapproximately 2.0 min. Furthermore, a pharmacokinetic model assuming twocompartments described the disappearance of cocaine from the brain. Theparameter estimates for t_(1/2α) and t_(1/2β) are 2.0 min and 14.5 min,respectively (FIG. 4B), values similar to those obtained for the plasma.

In the presence of 2E2, the peak cocaine concentration (˜490 ng/g) isobserved at the earliest sample time and it subsequently declinesrapidly over time (FIG. 4B). There is no indication of the normaldelayed influx and peak of the cocaine concentrations in the brain and asingle compartment model approximates the decline in cocaineconcentrations. The estimated t_(1/2) is 3.8 min, a value considerablyfaster than the t_(1/2β) value obtained in the absence of 2E2.Importantly, 2E2 produces an approximately 4.5-fold (78%) decrease inthe cocaine AUC in the brain.

Discussion

The low volume of distribution of 2E2 observed in mice is similar tothat previously reported for several murine and rat monoclonal IgG₁antibodies and human polyclonal IgG₁ antibodies in rats and isconsistent with 2E2's distribution being predominantly restricted to theblood volume. Additionally, the elimination t_(1/2) value for 2E2 isrelatively long and similar to that reported for other murine, rat andhuman antibodies in rats. This indicates that 2E2's effects on cocainepharmacokinetics could persist for several days after a singleinjection. Furthermore, the terminal elimination t_(1/2) of cocaine ismore than 400-fold faster than that of 2E2 and, therefore, it can beassumed that the plasma concentration of 2E2 is constant during thestudy of cocaine pharmacokinetics. Interestingly, although the V_(d) andt_(1/2) for 2E2 are similar to those previously described for antibodiesin rodents, there is no evidence for an initial distribution of 2E2 fromthe blood to the interstitial spaces.

As to 2E2's in vivo binding of cocaine, its effect on the plasmaconcentration of cocaine is saturable, which is consistent with thelimited number of cocaine molecules present. Furthermore, doses of 2E2that are only 10% to 30% of the dose of cocaine still provide ameasurable increase in plasma cocaine concentrations and a decrease inexposure of the brain to cocaine. This is consistent with reports that a0.3 molar ratio of anti-phencyclidine (PCP) Fabs decreases thebehavioral effects of PCP in rats. Furthermore, a 4 mg dose of ananti-cocaine mAb, representing a molar ratio of approximately 0.005, hasbeen reported to antagonize the behavioral effects of repeated 1 mg/kgdoses of cocaine HCl. While, 30 and 40 mg/kg doses of another murineanti-cocaine mAb decrease the self-administration of cocaine at molarratios of approximately 0.2 for each cocaine injection. The finding that2E2 produces a substantial reduction in the brain's exposure to cocaineat equimolar ratios and has measurable effectiveness at lower molarratios indicates that 2E2 will reduce brain concentrations even after amAb dose has been partially eliminated. Thus the efficacy of a givendose of 2E2 is prolonged.

The demonstration that an equimolar dose of a nonspecific antibody didnot significantly alter either plasma or brain concentrations of cocainerules out the possibility of nonspecific effects of infused IgG proteinsas an explanation for the mAb effects on cocaine pharmacokinetics.Therefore, the efficacy of anti-cocaine mAbs requires specificity of thebinding interaction between the drug/antibody molecules. However, thethree anti-cocaine mAbs with affinities ranging from very high(K_(d)=0.2 nM) to modest (K_(d)=40 nM, as measured in vitro atequilibrium) are approximately equipotent under the limited in vivoexperimental conditions tested. This suggests that the ability of anantibody to influence the pharmacokinetics of cocaine may not be highlyaffinity sensitive. Therefore, antibodies with a fairly broad range ofaffinities may have clinical efficacy. Antibodies with low affinity,that is having K_(d) s in the μM range, have been reported to amelioratesome behavioral effects of cocaine in rodents, but would most likely notbe as effective as 2E2 on treating cocaine-related disorders.

In the presence of 2E2, the initially observed approximately 10-foldincrease in the concentration of cocaine in plasma, the lack of aninitial distribution phase from the plasma and the reduction of the Vdssof cocaine to essentially that of 2E2 are all consistent with 2E2restricting cocaine's distribution predominantly to the blood volume.Therefore, the 2E2-induced decrease in brain cocaine concentrations isdue to an inhibition of cocaine distribution from the blood to thebrain. Furthermore, the reduction in the peak levels and thedistribution of cocaine to the brain occurs at all time points,indicating that 2E2 did not simply delay cocaine's distribution to thebrain. This report is the first to demonstrate that an anti-cocaine mAbcan prevent the entry of cocaine into the brain and it is consistentwith previous reports that active immunization-induced anti-cocaineantibodies decrease cocaine levels in brain after i.v., intranasal ori.p. cocaine administration. The ability of 2E2 to decrease brainconcentrations of cocaine is also consistent with the mAb-inducedreductions observed for other psychoactive drugs such as phencyclidine,methamphetamine and nicotine in rats.

The markedly altered distribution of cocaine is the result of cocainebinding to 2E2. While it may initially be believed that this mAb bindingof cocaine also restricts cocaine's access to the enzymes thatmetabolize it, thereby decreasing its clearance, there is no evidence ofan increase in the elimination t_(1/2) of cocaine in plasma. This isconsistent with the reported lack of effect of active immunization oneither the elimination of cocaine from plasma or on the rate ofmetabolism of nicotine. However, a murine anti-nicotine mAb and activeimmunization against nicotine have also been reported to significantlyincrease the elimination t_(1/2) of nicotine in rats. The reasons forthese discrepant results are not clear at present but do not appear tobe related to different affinities of the mAbs or the polyclonalantibodies for their target molecules. If anti-drug antibodies caninhibit the metabolism and slow the rate of drug elimination this wouldincrease the in vivo concentrations resulting from repeated drug dosesand may not be desirable for an immunotherapeutic agent. Importantly,the lack of effect of 2E2 on cocaine elimination from plasma shouldminimize the potential for 2E2 to become saturated following repeateddoses of cocaine.

In summary, the high affinity anti-cocaine mAb 2E2 limits thedistribution of cocaine to the plasma thus decreasing the levels ofcocaine reaching the brain without any detectable effect on the rate ofelimination of cocaine. The data further supports the general concept ofthe usefulness of immunotherapy for the treatment of drug abuse and isconsistent with mAb 2E2 being effective as a passive immunotherapy forthe prevention of relapse in cocaine abuse.

The foregoing description of various embodiments and principles of theinvention has been presented for the purposes of illustration anddescription. It is not intended to be exhaustive or to limit theinventions to the precise forms disclosed. Many alternatives,modifications, and variations will be apparent to those skilled the art.Moreover, although multiple inventive aspects and principles have beenpresented, these need not be utilized in combination, and variouscombinations of inventive aspects and principles are possible in lightof the various embodiments provided above. Accordingly, the abovedescription is intended to embrace all possible alternatives,modifications, aspects, combinations, principles, and variations thathave been discussed or suggested herein, as well as all others that fallwithin the principles, spirit and scope of the inventions as defined bythe claims.

1. A method for treating a cocaine-related disorder in an individual,comprising administering to the individual a therapeutic amount of anantibody comprising a human gamma heavy chain and a murine lambda lightchain.
 2. The method of claim 1, wherein the murine lambda light chainis partially murine derived.
 3. The method of claim 2, wherein thepartially murine lambda light chain comprises a murine variable regionand a human constant region.
 4. The method of claim 1, wherein themurine lambda light chain comprises SEQ ID NO: 1 or a derivativethereof.
 5. The method of claim 1, wherein the antibody binds cocaine ora derivative thereof.
 6. The method of claim 5, wherein the cocainederivative comprises at least one of cocaethylene and norcaine.
 7. Themethod of claim 1, wherein the antibody has at least a nanomolaraffinity for cocaine.
 8. The method of claim 1, wherein the antibody hasa K_(d) of about 40 nM or less.
 9. The method of claim 8, wherein theantibody has a K_(d) of about 4 nM.
 10. The method of claim 1, whereinthe cocaine-related disorder comprises at least one of overdose,addiction, dependence, and relapse.
 11. The method of claim 1, whereinthe human gamma heavy chain comprises SEQ ID NO: 2 or a derivativethereof.
 12. The method of claim 11, wherein the murine light chaincomprises SEQ ID NO: 1 or a derivative thereof.
 13. The method of claim1, wherein the antibody decreases the concentration of cocaine in thebrain of the individual.
 14. A method of treating a cocaine-relateddisorder in an individual comprising administering to the individual atherapeutic amount of an antibody comprising a human gamma heavy chainand a human kappa light chain at least partially derived from 1B3. 15.The method of claim 14, wherein the human kappa light chain comprises atleast one of SEQ ID NO: 3, SEQ ID NO: 14, or a derivative thereof.
 16. Amonoclonal antibody comprising a human gamma heavy chain and a murinelambda light chain.
 17. The monoclonal antibody of claim 16, wherein thelight chain comprises SEQ ID NO: 1 or a derivative thereof.
 18. Themonoclonal antibody of claim 16, wherein the human gamma heavy chaincomprises SEQ ID NO: 2 or a derivative thereof.
 19. A monoclonalantibody comprising a human gamma heavy chain and a human kappa lightchain at least partially derived from 1B3.
 20. The antibody of claim 18,wherein the human gamma heavy chain comprises SEQ ID NO: 2 or aderivative thereof and the human kappa light chain comprises at leastone of SEQ ID NO: 3, SEQ ID NO: 14 or a derivative thereof.
 21. A methodfor binding cocaine or a derivative thereof comprising contactingcocaine or a derivative thereof with an effective amount of an antibody,wherein the antibody comprises a human gamma heavy chain and a lightchain, wherein the light chain is selected from the group consisting of:a murine lambda light chain, a human kappa light chain derived at leastpartially from 1B3, and combinations thereof.